Effects of free radicals and amyloid beta protein on the currents of expressed rat receptors in Xenopus oocytes

Objective To investigate the effects of free radicals (FRs) and amyloid bet a protein 1-40 (A beta (1-40)) on the functions of expressed neurotransmitt er receptors (NRs) in Xenopus oocytes. Methods Total RNA and messenger RNA (mRNA) was prepared from 3-month-old Wi star rat brain tissues with Promega kits and microinjected into maturated X enopus oocytes (stages V-VI) with 50 nl (50 ng) for each oocyte. The microi njected oocytes were incubated with modified Bath's solution at 19.0 degree sC+/-1.0 degreesC for receptor expression and their currents were recorded with double electrode voltage clamp technique. Superoxide anion free radica ls (SAFRs) were produced via a reaction system (HPX/XO) with hypoxanthine ( HPX, 0.05 mol/L) and xanthine oxidase (XO, 0.1 U/L). In order to observe th e effects of A beta and SAFRs on the expressed glutamate receptor, HPX/XO a nd A beta (1-40) were added to incubation solution at 12 h, 24 h and 96 h b efore recording. Results The results showed that the oocytes expressed functional NRs origin ating from rat brain tissues. These NRs included muscarinic acetylcholine ( mACh), glutamate (Glu), dopamine (DA), serotonin (5-HT) and gamma -aminobut yric acid (GABA). The current characteristics of expressed receptors were i nward currents carried by chloride ion with their equibrilium potentials cl ose to -22 mV. The extent of effect on the current of expressed glutamate r eceptor from rat brain was different among different A beta concentrations and incubation times. A beta (1-40) at a concentration of 20 nmol/L had lit tle effect on the currents of expressed rat brain glutamate receptors up to 24 h of incubation period; but the currents of glutamate receptor were sig nificantly decreased (25% off, P < 0.01) in the treatment of 60 nmol/L A<be ta>(1-40) over 24 h. Moreover, when 20 nmol/L A beta (1-40) was co-incubate d over 12 h with SAFRs produced by the reaction system of HPX/XO, it was fo und that the currents of expressed rat brain glutamate receptors had been c hanged markedly. When the oocytes were co-treated with 60 nmol/L A beta (1- 40) and SAFRs over a period of 12 h, the currents of glutamate receptor sig nificantly decreased (21% off, P < 0.05), and the decreased percentage reac hed 52% over 24 h co-treatment with 60 nmol/L A<beta>(1-40) and SAFRs. In a ddition, vitamin E had a partial effect against this inhibitory effect. Conclusion The results suggest that A beta has a kind of inhibitory effect upon the current of the glutamate receptor, similar to the effects of free radicals. The effects can be antagonized by vitamin E. These imply that A b eta may play a role via inhibiting receptor function in the pathophysiology of Alzheimer's disease.